The present invention relates in general to methods and kits for performing nucleic acid hybridization assays and in particular to methods and kits for immobilizing a target nucleic acid on a solid support
One characteristic property of nucleic acid, which forms the heritable material of all living organisms, is its ability to form sequence-specific hydrogen bonds with a nucleic acid having a complementary sequence of nucleotides. This ability of nucleic acids to form sequence-specific hydrogen bonds (i.e., to hybridize) with complementary strands of nucleic acid is exploited in techniques generally called hybridization assays.
In a hybridization assay, a nucleic acid having a known sequence is used as a "probe" to search a sample for a "target" complementary sequence. Labelling of the hybrid formed by the probe and the target permits the detection and quantitation of target complementary sequence in the sample.
Because all strains of a particular microorganism share a genetic component in the form of nucleic acids susceptible to diagnosis by means of a hybridization assay, such hybridization assays are valuable research and medical tools. Detection of specific target nucleic acids enables accurate diagnosis of bacterial, fungal and viral disease states in humans, animals and plants. Additionally, the ability to probe for a specific nucleotide sequence is of potential use in the identification and diagnosis of human genetic disorders.
In one type of hybridization assay, called solution hybridization, a labelled probe nucleic acid is added to a solution of a sample to be searched for a target nucleic acid. In order to ensure that both the probe and a target are in a single-stranded state suitable for hybridization, the sample and probe are heated in order to break (denature) the hydrogen bonds which are found between complementary strands of a double-stranded probe or a double-stranded target, or which are found within secondary structure of a probe or target. Upon cooling, the reaction is reversed and double-stranded nucleic acid is allowed to form. The amount of double-stranded nucleic acid which forms may be determined by scintillation counting of the label on the probe after degradation of unhybridized single strands or after isolating double-stranded DNA by passing the hybridization solution over a hydroxyapatite column which selectively retains the double-stranded form. However, if either the probe or the target was introduced in double-stranded form, a reaction reforming (renaturing) double-stranded probe or a double-stranded target competes with the hybridization reaction between probe and target and thereby reduces the sensitivity of this technique.
In another approach to hybridization assays, the renaturation problem is circumvented by immobilizing denatured target nucleic acid on a support. Retention of a labelled probe on a support-bound target after passage of the support-bound target through a solution containing the probe permits detection and quantitation of the target by measurement of the amount of bound label. See, e.g., Falkow, et al., U.S. Pat. No. 4,358,535; and Shafritz, European Patent Application No. A1-0062286. Nevertheless, because the amount of labelled probe is far in excess of the amount of target present, non-specific binding of the labelled probe to the support may swamp the detectable signal from a target present in small amounts.
Still another approach to hybridization assays is called a "sandwich" hybridization. A two-step sandwich hybridization procedure involves the use of an immobilized target nucleic acid which is exposed in a first step to a first nucleic acid probe having a first portion complementary to the target and having a second portion which is not complementary to the target. In a second step, a second, labelled nucleic acid probe, which is complementary to the second portion of the first probe, is allowed to hybridize to the first probe, forming a "sandwich" comprising the first probe between the target and the second probe. Dunn, et al., Cell, 12: 23-36 (1977). The sandwich hybridization procedure is relatively easy to perform and is not seriously affected by protein or other biological contaminants. Ranki, et al., Gene, 21: 77-85 (1983). However, a two-step sandwich hybridization assay involves considerable delay associated with immobilization of the sample on a filter.
A one-step sandwich assay involves the use of a first nucleic acid probe immobilized on a filter. The first nucleic acid probe is complementary to a first portion of a target nucleic acid. In a single step the filter-bound first probe is exposed to a sample to be searched for the target nucleic acid sequence and to a second, labelled nucleic acid probe complementary to a second portion of the target nucleic acid which portion is separate from (i.e., non-overlapping with) the portion of the target to which the first probe is complementary. Ranki, et al., U.S. Pat. No. 4,486,539. This one-step technique eliminates the delay caused by immobilization of a sample on a filter; eliminates differences between the types of treatment required for binding ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) to certain types of support inasmuch as the first probe may be selected to suit the support; and is even less sensitive to contaminating materials in the sample, e.g., mucus, than is a direct hybridization assay where the target is bound to the support. Ranki, et al., Curr.Top. Microbiol.Immunol., 104: 307-318 (1983). Nevertheless, leakage of the first probe from the support during hybridization occurs frequently and significantly diminishes the sensitivity of the assay. Because hybridization occurs more readily where both partners to the hybridization are in solution rather than where either is bound to a support, the target preferentially binds to leaked first probe. Inasmuch as the amount of leaked first probe is likely to be in excess of the amount of target present, a significant amount of target nucleic acid may be consumed by the leaked probe rather than be bound to the support.
In another approach to hybridization, called blot hybridization, nucleic acids within a sample are separated according to size by electrophoresis through a gel and are transferred to a nitrocellulose filter on which they are immobilized in their relative positions on the gel. Because any target in the sample is confined to a distinct band on the filter, even weak signals resulting from small amounts of target may be distinguished from non-specific background after exposure to a radiolabelled probe. Bornkamm, et al., Curr.Top.Microbiol. Immunol., 104: 288-298 (1983). Nevertheless, the added difficulty and expense of performing an electrophoretic separation on a sample limits the practicality of applying a blot hybridization technique in a clinical setting.
Where a sample is in the form of a touch smear of a fluid, a section through cells, or chromosomal squashes from cells on slides, hybridization may be performed in situ. Generally, a radioactively labelled probe is applied to the sample which is bound to the slide in a histological preparation. After coating the slide with a photographic emulsion, autoradiographic procedures reveal the location of target-probe hybrids by means of clusters of silver grains formed in the emulsion over the hybridization site. However, where only a few grains are observed, it is difficult if not impossible to prove that the hybridization is specific. Bornkamm, et al., supra.
An attempt has been made to improve upon the in situ hybridization technique by using as a label albumin-coated gold spheres which are cross-linked to a nucleic acid having a poly(dT) tail. Chromosomes are hybridized in situ with a probe having a poly(dA) tail which is in turn hybridized to the poly(dT) tail attached to the gold sphere in order to mark the site of hybridization. Wu, et al., Proc.Natl.Acad.Sci. (USA), 78: 7059-7063 (1981). Despite having advantages over other in situ hybridization techniques, this procedure shares a disadvantage with the other in situ hybridization methods in that it requires microscopic examination of large numbers of slides on which is spread only a small amount of sample. As a result, this approach is difficult and time consuming to apply in a clinical setting.
Thus there exists a continuing need in the art for improved nucleic acid hybridization methods for minimizing the effect of probe leakage on the accurate detection of target molecules in a sample.